In several areas of technology it is desirable to separate polymeric compounds on the basis of their size, configuration, charge or other fundamental characteristics. For example, techniques relating to molecular biology and biotechnology frequently involve the analysis of a mixture of polypeptides or polynucleotides, which may be separated in accordance with their relative sizes. Results can provide indication of the size and relative abundance of compounds in the mixture with significant accuracy. Indeed, some techniques enable the separation of polynucleotides with a resolution of a single nucleotide, which is critical for analysis such as DNA sequencing.
Traditionally, compounds such as polypeptides and polynucleotides are separated by electrophoresis involving the application of an electric current through a buffered solution containing the compounds. During the electrophoresis the compounds may be forced to migrate through a matrix material that hinders progression of the migration. Such matrix materials may include agarose or polyacrylamide. Longer polymeric compounds migrate more slowly through the matrix when compared to shorter polymeric compounds, resulting in fairly rapid separation of the compounds on the basis of polymer length.
More recently, much attention has been focused on the free-solution electrophoresis of charged-uncharged polymer conjugates in microchannel electrophoresis systems such as capillary electrophoresis or microchip electrophoresis systems. The performance of electrophoresis in free solution overcomes the need for gels or entangled polymer solutions for the electrophoretic separation of polyelectrolytes, while offering a means for the molar mass profiling of uncharged polymers. End-labeled free-solution electrophoresis (ELFSE), for instance, was successfully used to sequence ssDNA up to 110 bases in less than 20 minutes [1]. This technique cleverly uses an uncharged “label” or “drag” molecule attached to each single-stranded DNA (ssDNA) chain in order to break the local balancing between friction and electric force [2, 3, 4, 5, 6] which normally leads to co-migration of all ssDNA lengths [7, 8] (excepting very small fragments [9, 10]) in free solution. More recently, a complementary technique called free solution conjugate electrophoresis (FSCE) has been used to characterize uncharged, water-soluble polymers that can be uniquely conjugated to ssDNA [11, 12, 13]. Here the ssDNA chains are of uniform length, and act as engines to pull the varying lengths of uncharged polymers for electrophoresis leading to single-monomer resolution over a wide range of molecular sizes. In fact, the resolution obtained was approximately five times higher, and the separation efficiencies were increased by 150% compared to the more traditional RP-HPLC [12]. For both FSCE and ELFSE, the theoretical equation utilized for the overall mobility μ of the charged-uncharged block copolymer was a uniformly weighted average [5, 6. 11, 13]:
                    μ        =                                            μ              0                        ⁢                                          M                c                            N                                =                                    μ              0                        ⁢                                          M                c                                                              M                  c                                +                                                      α                    1                                    ⁢                                      M                    u                                                                                                          (        1        )            where Mc is the number of charged monomers each of mobility μc, and Mu is the number of uncharged monomers. This equation comes from a pioneer investigation of Long and co-workers into the electrophoresis of polymers containing both charged and uncharged monomers [14]. The factor α1 rescales Mu account for the difference in hydrodynamic properties arising for example from the different persistence lengths (a measure of flexibility) of the charged and uncharged polymers. Hence the α1 value depends on the chemistry of the molecules and varies with both temperature and buffer ionic strength (which affect the molecules' flexibilities). In fact, α=α1Mu enables a counting of uncharged units which have the same friction as one ssDNA monomer, such that the total number of effective monomers is N=Mc+α1Mu. The α1 value is an important determinant of the mobility since the frictional drag of the uncharged polymer is what selectively slows down longer conjugates in FSCE, and determines the read length of ELFSE.
Therefore, it is generally known in the art that the modification of polynucleotides for example by the covalent attachment of selected moieties can increase the frictional ‘drag’ of the polynucleotide during free-solution electrophoresis.
The work of Long and coworkers, as well as the work of others, has increased our general understanding of the mechanisms of polymeric compound separation by free solution electrophoresis. Moreover, the use of tags to alter the frictional drag characteristics of oligonucleotides during free-solution electrophoresis has provided improvements in these techniques. Nonetheless, there remains a continuing need to develop methods for the separation of polymeric compounds that are simple, effective, and rapid. In particular there is a need to develop methods for the separation of polymeric compounds such as polypeptides or polynucleotides with a high level of accuracy and a resolution of a single amino acid or nucleotide.